The present invention related generally to novel molecules and more particularly novel proteinaceous molecules involved in or associated with regulation of cell activities and/or viability. The present invention is particularly directed to novel serene proteinases and a novel kinase and to derivatives, agonists and antagonists thereof. In one embodiment, the present invention provides a novel serine proteinase, referred to herein as xe2x80x9cHELA2xe2x80x9d or xe2x80x9ctestisinxe2x80x9d, which has roles in spermatogenesis, in suppressing testicular cancer and as a marker for cancers.
The rapidly increasing sophistication of recombinant DNA technology is greatly facilitating research and development in the medical and allied health fields. This is particularly the case in the area of cell regulation leading to a greater understanding of the events leading to or involved in cancer, development of acquired immunodeficiency disease syndrome (AIDS), neurological disorders, heart disease, tissue graft rejection and infertility amongst many other conditions.
Two particularly important classes of molecules are the proteinases and kinases.
Proteinases play important roles in a number of physiological and pathological processes such as proteolytic cascades involved in blood coagulation, fibrinolysis and complement activation as well as cleavage of growth factors, hormones and receptors, the release of bioactive molecules and processes involving cell proliferation and development, inflammation, tumour growth and metastasis. Of particular significance are the cellular proteinases, or those proteinases synthesized in cells and tissues which serve to activate or deactivate proteins responsible for performing specific functions. These proteinases may be found outside the cell, within the cell or may be present on the cell surface.
Serine proteinases are particularly important. These proteinases are characterised by a mechanism involving serine, histidine and aspartate amino acids in the serine proteinase active site. Members of the serine proteinase family which play important roles in a range of cellular functions and which have demonstrated causative roles in human diseases include tissue-type plasminogen activator and thrombin (thrombosis and blood clotting), urokinase-type plasminogen activator (cancer and metastasis), trypsin and elastase (emphysema and liver disease) and angiotensin converting enzyme (hypertension).
A serine proteinase is also implicated in TNFxcex1 degradation and soluble TNF-receptor (p75) release by THP1 cells (Vey et al. Eur. J. Imm. 26, 2404-2409, 1996). Serine proteinases have been implicated in the activation of macrophages (Nakabo et al. J. Leukocyte Biol. 60, 328-336, 1996), in nuclear laminin degradation in apoptosis (McConkey et al. J.Biol. Chem., 271, 22398-22406, 1996), in prostaglandin-E2 induced release of soluble TNF receptor shedding (Choi et al. Cellular Immunology 170, 178-184, 1996), in PAF synthesis (Bussolino et al. Eur. J. Immunol. 24, 3131-3139, 1994), and in the proteolysis of IkB, a regulatory molecule important in signal transduction and apoptosis. Release of serine proteinases known as granzymes is central to CTL killing and many of the substrates cleaved by granzymes are also cleaved by cellular proteinases (for example, IL-1xcex2 is a substrate for Granzyme B as well as the cysteine proteinase, interleukin 1xcex2-converting enzyme (ICE)). Granzyme A, a serine proteinase with Arg-amidolytic activity, has been reported to induce the production of IL-6 and IL-8 in lung fibroblasts (Sower et al. Cellular Immunology 171, 159-163, 1996) and cleaves IL1xcex2 to a 17 kD mature form that is biologically active.
Kinases are a large group of molecules, many of which regulate the response of cells to external stimuli. These molecules regulate proliferation and differentiation in eukaryotic cells frequently via signal transduction pathways.
The identification of new serine proteinases and kinases permits the development of a range of derivatives, agonists and antagonists at the nucleic acid and protein levels which in turn have applications in the treatment and diagnosis of a range of conditions such as cancer, inflammation, neurological disorders amongst many other conditions including conditions which initiate or promote apoptosis such as viral infection, old age and drug abuse. One particularly useful serine proteinase HELA2 (testisin) identified in accordance with the present invention is involved in spermatogenesis, testicular cancer and as a marker for cancer.
Sequence Identity Numbers (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the specification are defined at the end of the subject specification.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
One aspect of the present invention provides a novel molecule in isolated form involved in or associated with regulation of cell activity and/or viability.
Another aspect of the present invention contemplates an isolated proteinaceous molecule involved in or associated with regulation of cell activity and/or viability comprising a sequence of amino acids encoded by a nucleotide sequence, at least a portion of which, is capable of being amplified by polymerase chain reaction (PCR) using the following primers:
5xe2x80x2 ACAGAATTCTGGGTIGTIACIGCIGCICAYTG3xe2x80x2 [SEQ ID NO:1]; and
5xe2x80x2 ACAGAATTCAXIGGICCICCIC/GT/AXTCICC3xe2x80x2 [SEQ ID NO:2];
or a complementary form of said primers.
The proteinaceous molecule of the present invention may be a serine proteinase or a kinase.
Yet another aspect of the present invention is directed to an isolated serine proteinase comprising the amino acid sequence substantially set forth in SEQ ID NO:4 or an amino acid sequence having at least about 50% similarity to all or part thereof. This serine proteinase is referred to herein as a short isoform (S) of xe2x80x9cHELA2xe2x80x9d or xe2x80x9ctestisinxe2x80x9d.
Still another aspect of the present invention relates to an isolated serine proteinase comprising the amino acid sequence substantially as set forth in SEQ ID NO:6 or an amino acid sequence having at least about 50% similarity to all or part thereof. This serine proteinase is referred to herein as a long isoform (L) of HELA2 (testisin).
Still yet another aspect of the present invention provides an isolated serine proteinase comprising an amino acid sequence substantially as set forth in SEQ ID NO:8 or an amino acid sequence having at least about 50% similarity to all or part thereof. This serine proteinase is referred to herein as xe2x80x9cATC2xe2x80x9d.
Even yet another aspect of the present invention is directed to a serine proteinase in isolated form comprising a sequence of amino acids encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:3 or a nucleotide sequence having at least 50% similarity to all or part thereof or a nucleotide sequence capable of hybridising to the sequence set forth in SEQ ID NO:3 under low stringency conditions at 42xc2x0 C.
Another aspect of the present invention relates to a serine proteinase in isolated form comprising a sequence of amino acids encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:5 or a nucleotide sequence having at least 50% similarity to all or part thereof or a nucleotide sequence capable of hybridising to the sequence set forth in SEQ ID NO:5 under low stringency conditions at 42xc2x0 C.
Still another aspect of the present invention provides a serine proteinase in isolated form comprising a sequence of amino acids encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:7 or a nucleotide sequence having at least 50% similarity to all or part thereof or a nucleotide sequence capable of hybridising to the sequence set forth in SEQ ID NO:7 under low stringency conditions at 42xc2x0 C.
Another embodiment of the present invention is directed to a kinase in isolated form comprising an amino acid sequence substantially as set forth in SEQ ID NO:10 or having 50% amino acid similarity to all or part thereof. This kinase is referred to herein as xe2x80x9cBCON3xe2x80x9d.
In a related embodiment, the kinase comprises an amino acid sequence encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:9 or a nucleotide sequence having at least 50% similarity to all or part of the nucleotide sequence set forth in SEQ ID NO:9 or a nucleotide sequence capable of hybridising to the nucleotide sequence set forth in SEQ ID NO:9 under low stringency conditions at 42xc2x0 C.
The present invention further provides an isolated nucleic acid molecule encoding a polypeptide wherein at least a portion of said nucleic acid molecule is capable of being amplified by polymerase chain reaction (PCR) using the following primers:
5xe2x80x2 ACAGAATTCTGGGTIGTIACIGCIGCICAYTG3xe2x80x2 [SEQ ID NO:1]; and
5xe2x80x2ACAGAATTCAXIGGICCICCIC/GT/AXTCICC3xe2x80x2 [SEQ ID NO:2];
or a complementary form of said primers.
The present invention also provides an isolated nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:3 or having 50% similarity to all or part thereof or a nucleic acid molecule capable of hybridising to SEQ ID NO:3 under low stringency conditions at 42xc2x0 C.
Another aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:5 or having 50% similarity to all or part thereof or a nucleic acid molecule capable of hybridising to SEQ ID NO:5 under low stringency conditions at 42xc2x0 C.
Still another aspect of the present invention is directed to an isolated nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:7 or having 50% similarity to all or part thereof or a nucleic acid molecule capable of hybridising to SEQ ID NO:7 under low stringency conditions at 42xc2x0 C.
Even still another aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:9 or having 50% similarity to all or part thereof or a nucleic acid molecule capable of hybridising to SEQ ID NO:9 under low stringency conditions at 42xc2x0 C.
Another aspect of the present invention provides an isolated serine proteinase encoded by a gene proximal to a cluster of genes on a mammalian chromosome.
More particularly, this aspect of the present invention is directed to a serine proteinase encoded by a gene proximal to a cluster of genes or human chromosome 16p13.3 or its equivalent in a non-human species.
Still more particularly, the serine proteinase is encoded by a gene comprising a nucleotide sequence substantially as set forth in SEQ ID NO:3 or 5 or 28 or 29 or 30 or a nucleotide sequence having at least 50% similarity to any one thereof or a nucleotide sequence capable of hybridizing to any one of SEQ ID NO:3 or 5 or 28 or 29 or 30 under low stringency conditions at 42xc2x0 C. or a nucleotide sequence encoding a serine proteinase having an amino acid sequence substantially as set forth in SEQ ID NO:4 or 6 or an amino acid sequence having at least about 50% similarity to SEQ ID NO:4 or 6.